Male contraceptive compositions and methods of use
09789120 · 2017-10-17
Assignee
Inventors
Cpc classification
A61K31/5517
HUMAN NECESSITIES
A61P15/00
HUMAN NECESSITIES
International classification
A01N43/00
HUMAN NECESSITIES
A61K31/554
HUMAN NECESSITIES
A61K31/553
HUMAN NECESSITIES
Abstract
The invention relates to compositions and methods for effecting male contraception.
Claims
1. A method of reducing or inhibiting spermatogenesis in a healthy fertile male subject, the method comprising administering an effective amount of a compound or a salt thereof that inhibits a bromodomain testis-specific protein (BRDT) to the male subject, wherein the compound is of Formula (I): ##STR00444## wherein: X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, aryl optionally substituted with alkyl, halogen, amino, or —OH, or heteroaryl optionally substituted with alkyl, halogen, amino, or —OH; R.sub.2 is H, D (deuterium), halogen, or alkyl optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; (iv) N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
2. A method of reducing the rate of male fertility in a healthy fertile male subject, the method comprising administering an effective amount of a compound or a salt thereof that inhibits a BRDT to the male subject, wherein the compound is of Formula (I): ##STR00445## wherein: X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, aryl optionally substituted with alkyl, halogen, amino, or —OH, or heteroaryl optionally substituted with alkyl, halogen, amino, or —OH; R.sub.2 is H, D (deuterium), halogen, or alkyl optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; (iv) N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
3. The method of claim 1, wherein the method comprises administering the compound or a salt thereof in an amount sufficient to reduce sperm number and/or reduce sperm motility.
4. The method of claim 1, wherein the method comprises administering the compound or a salt thereof in an amount sufficient to induce azoospermia, oligozoospermia, and/or asthenozoospermia.
5. The method of claim 4, wherein the method induces a contraceptive effect in the subject.
6. The method of claim 1, wherein the compound or a salt thereof is administered to the subject orally, transdermally, or by injection.
7. The method of claim 6, wherein the compound or a salt thereof is administered in the form of a tablet or capsule.
8. The method of claim 6, wherein the compound or a salt thereof is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.
9. The method of claim 1, wherein the compound or a salt thereof is administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
10. The method of claim 1, wherein the compound is JQ1 or a compound of Formula II: ##STR00446## wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, each of which wherein alkyl is optionally substituted with halogen or —OH; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; R′.sub.1 is H, —COO—R.sub.3, —CO—R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, or heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; m is 0, 1, 2, or 3, or salt thereof; wherein the compound contains a fluorine or chlorine atom.
11. The method of claim 10, wherein the subject is human.
12. The method of claim 2, wherein the method comprises administering the compound or a salt thereof in an amount sufficient to reduce sperm number and/or reduce sperm motility.
13. The method of claim 2, wherein the method comprises administering the compound or a salt thereof in an amount sufficient to induce azoospermia, oligozoospermia, and/or asthenozoospermia.
14. The method of claim 13, wherein the method induces a contraceptive effect in the subject.
15. The method of claim 2, wherein the compound or a salt thereof is administered to the subject orally, transdermally, or by injection.
16. The method of claim 15, wherein the compound or a salt thereof is administered in the form of a tablet or capsule.
17. The method of claim 15, wherein the compound or a salt thereof is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.
18. The method of claim 2, wherein the compound or a salt thereof is administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
19. The method of claim 2, wherein the compound is JQ1 or a compound of Formula (I): ##STR00447## wherein: X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, aryl optionally substituted with alkyl, halogen, amino, or —OH, or heteroaryl optionally substituted with alkyl, halogen, amino, or —OH; R.sub.2 is H, D (deuterium), halogen, or alkyl optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; (iv) N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; or a salt thereof; wherein the compound contains a fluorine or chlorine atom.
20. The method of claim 2, wherein the subject is human.
21. The method of claim 1, wherein the compound is JQ1 or a compound of Formula III: ##STR00448## wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; and (iv) NH.sub.2, N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; and m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
22. The method of claim 1, wherein the compound is JQ1 or a compound of Formula IV: ##STR00449## wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, aryl optionally substituted with alkyl, halogen, amino, or —OH, or heteroaryl optionally substituted with alkyl, halogen, amino, or —OH; R.sub.2 is H, D (deuterium), halogen, or alkyl optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; and (iv) N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; and m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
23. The method of claim 1, wherein the compound is JQ1 or a compound of Formulae V-XXII: ##STR00450## ##STR00451## ##STR00452## ##STR00453## wherein: R is H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl, —C.sub.2-C.sub.8 alkynyl, —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or aryl; R′ is H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl, —C.sub.2-C.sub.8 alkynyl, —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, amino, or —OH; and X is a substituent of an aryl group; wherein the compound contains a fluorine or chlorine atom.
24. The method of claim 2, wherein the compound is JQ1 or a compound of Formula III: ##STR00454## wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted with halogen, alkyl, amino, —OH, or aryl; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; and (iv) NH.sub.2, N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; and m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
25. The method of claim 2, wherein the compound is JQ1 or a compound of Formula IV: ##STR00455## wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, wherein alkyl is optionally substituted with halogen or —OH; ring A is phenyl or thienyl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, amino, or —OH; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, aryl optionally substituted with alkyl, halogen, amino, or —OH, or heteroaryl optionally substituted with alkyl, halogen, amino, or —OH; R.sub.2 is H, D (deuterium), halogen, or alkyl optionally substituted with halogen, amino, or —OH; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, or heteroaryl; (ii) heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or heterocyclyl; and (iv) N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted with halogen, alkyl, amino, —B(OH).sub.2, —SO.sub.2Na, or —OH; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; and m is 0, 1, 2, or 3; wherein the compound contains a fluorine or chlorine atom.
26. The method of claim 2, wherein the compound is JQ1 or a compound of Formulae V-XXII: ##STR00456## ##STR00457## ##STR00458## ##STR00459## wherein: R is H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl, —C.sub.2-C.sub.8 alkynyl, —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, alkyl, amino, —OH, or aryl; R′ is H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl, —C.sub.2-C.sub.8 alkynyl, —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted with halogen, amino, or —OH; and X is a substituent of an aryl group; Y is C; and n is 1 in Formula (V); wherein the compound contains a fluorine or chlorine atom.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(10) This invention is based, at least in part, on the discovery that a small-molecule inhibitor (JQ1) of the bromodomain and extra-terminal (BET) subfamily of epigenetic reader proteins is essential for chromatin remodeling during spermiogenesis. Biochemical analysis confirms that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. The invention is also based on the discovery that treatment of mice with JQ1 reduced the number and motility of spermatozoa, as well as testis size. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte stage cause a dramatic decrease in fertilized oocytes and a reversible contraceptive effect in males. Accordingly, the present invention is directed to a novel type of male contraceptive that can cross the blood:testis boundary and inhibits bromodomain activity during spermatogenesis.
(11) Bromodomain-Containing Proteins
(12) Gene regulation is fundamentally governed by reversible, non-covalent assembly of macromolecules. Signal transduction to RNA polymerase requires higher-ordered protein complexes, spatially regulated by assembly factors capable of interpreting the post-translational modification states of chromatin. Epigenetic readers are structurally diverse proteins each possessing one or more evolutionarily conserved effector modules, which recognize covalent modifications of histone proteins or DNA. The ε-N-acetylation of lysine residues (Kac) on histone tails is associated with an open chromatin architecture and transcriptional activation.sup.3. Context-specific, molecular recognition of acetyl-lysine is principally mediated by bromodomains.
(13) Bromodomain-containing proteins are of substantial biological interest, as components of transcription factor complexes (TAF1, PCAF, Gen5 and CBP) and determinants of epigenetic memory.sup.4. There are 41 human proteins containing a total of 57 diverse bromodomains. Despite large sequence variations, all bromodomains share a conserved fold comprising a left-handed bundle of four alpha helices (α.sub.Z, α.sub.A, α.sub.B, α.sub.C), linked by diverse loop regions (ZA and BC loops) that determine substrate specificity. Co-crystal structures with peptidic substrates showed that the acetyl-lysine is recognized by a central hydrophobic cavity and is anchored by a hydrogen bond with an asparagine residue present in most bromodomains.sup.5. The bromodomain and extra-terminal (BET)-family (BRD2, BRD3, BRD4 and BRDT) shares a common domain architecture comprising two N-terminal bromodomains that exhibit high level of sequence conservation, and a more divergent C-terminal recruitment domain.sup.6.
(14) The invention features compositions and methods that are useful for inhibiting human bromodomain proteins.
(15) Compounds of the Invention
(16) The invention provides compounds (e.g., JQ1 and compounds of formulas delineated herein) that bind in the binding pocket of the apo crystal structure of the first bromodomain of a BET family member (e.g., BRDT, BRD2, BRD3, BRD4). Without wishing to be bound by theory, these compounds are particularly effective in reducing male fertility. In one approach, compounds useful for reducing male fertility are selected using a molecular docking program to identify compounds that are expected to bind to a bromodomain structural binding pocket. In certain embodiments, a compound of the invention can prevent, inhibit, or disrupt, or reduce by at least 10%, 25%, 50%, 75%, or 100% the biological activity of a BET family member (e.g., BRD2, BRD3, BRD4, BRDT) and/or disrupt the subcellular localization of such proteins, e.g., by binding to a binding site in a bromodomain apo binding pocket.
(17) In certain embodiments, a compound of the invention is a small molecule having a molecular weight less than about 1000 daltons, less than 800, less than 600, less than 500, less than 400, or less than about 300 daltons. Examples of compounds of the invention include JQ1 and other compounds that bind the binding pocket of the apo crystal structure of the first bromodomain of a BET family member (e.g., BRD4 (hereafter referred to as BRD4(1); PDB ID 2OSS). JQ1 is a novel thieno-triazolo-1,4-diazepine. The invention further provides pharmaceutically acceptable salts of such compounds.
(18) In one aspect, the compound is a compound of Formula I:
(19) ##STR00001##
wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, each of which is optionally substituted; ring A is aryl or heteroaryl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; each of which is optionally substituted; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl; R.sub.2 is H, D (deuterium), halogen, or optionally substituted alkyl; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted; and (iv) NH.sub.2, N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; provided that (a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 1 and L is —CO—N(R.sub.3R.sub.4), then R.sub.3 and R.sub.4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; (b) if ring A is thienyl, X is N, R is substituted phenyl, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 1 and L is —CO—N(R.sub.3R.sub.4), and one of R.sub.3 and R.sub.4 is H, then the other of R.sub.3 and R.sub.4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and (c) if ring A is thienyl, X is N, R is substituted phenyl, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 1 and L is —COO—R.sub.3, then R.sub.3 is not methyl or ethyl; or a salt, solvate or hydrate thereof.
(20) In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted.
(21) In certain embodiments, L is H, —COO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3 or optionally substituted aryl. In certain embodiments, each R.sub.3 is independently selected from the group consisting of: H, —C.sub.1-C.sub.8 alkyl, containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; or NH.sub.2, N═CR.sub.4R.sub.6.
(22) In certain embodiments, R.sub.2 is H, D, halogen or methyl.
(23) In certain embodiments, R.sub.B is alkyl, hydroxyalkyl, haloalkyl, or alkoxy; each of which is optionally substituted.
(24) In certain embodiments, R.sub.B is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH.sub.2OC(O)CH.sub.3.
(25) In certain embodiments, ring A is a 5 or 6-membered aryl or heteroaryl. In certain embodiments, ring A is thiofuranyl, phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl.
(26) In certain embodiments, ring A is phenyl or thienyl.
(27) In certain embodiments, m is 1 or 2, and at least one occurrence of R.sub.A is methyl.
(28) In certain embodiments, each R.sub.A is independently H, an optionally substituted alkyl, or any two R.sub.A together with the atoms to which each is attached, can form an aryl.
(29) In another aspect, the compound is a compound of Formula II:
(30) ##STR00002##
wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, each of which is optionally substituted; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R′.sub.1 is H, —COO—R.sub.3, —CO—R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl; —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl; each of which may be optionally substituted; m is 0, 1, 2, or 3; provided that if R′.sub.1 is —COO—R.sub.3, X is N, R is substituted phenyl, and R.sub.B is methyl, then R.sub.3 is not methyl or ethyl; or a salt, solvate or hydrate thereof.
(31) In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted. In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl.
(32) In certain embodiments, R′.sub.1 is —COO—R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl; and R.sub.3 is —C.sub.1-C.sub.8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted. In certain embodiments, R′.sub.1 is —COO—R.sub.3, and R.sub.3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or R′.sub.1 is H or optionally substituted phenyl.
(33) In certain embodiments, R.sub.B is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH.sub.2OC(O)CH.sub.3.
(34) In certain embodiments, R.sub.B is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, or COOCH.sub.2OC(O)CH.sub.3.
(35) In certain embodiments, each R.sub.A is independently an optionally substituted alkyl, or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl.
(36) In certain embodiments, each R.sub.A is methyl.
(37) In another aspect, the compound is a compound of formula III:
(38) ##STR00003##
wherein X is N or CR.sub.5; R.sub.5 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, each of which is optionally substituted; ring A is aryl or heteroaryl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R is alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted; and (iv) NH.sub.2, N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; provided that: (a) if ring A is thienyl, X is N, R is phenyl or substituted phenyl, R.sub.B is methyl, then R.sub.3 and R.sub.4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; and (b) if ring A is thienyl, X is N, R is substituted phenyl, R.sub.2 is H, R.sub.B is methyl, and one of R.sub.3 and R.sub.4 is H, then the other of R.sub.3 and R.sub.4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and or a salt, solvate or hydrate thereof.
(39) In certain embodiments, R is aryl or heteroaryl, each of which is optionally substituted. In certain embodiments, R is phenyl or pyridyl, each of which is optionally substituted.
(40) In certain embodiments, R is p-Cl-phenyl, o-Cl-phenyl, m-Cl-phenyl, p-F-phenyl, o-F-phenyl, m-F-phenyl or pyridinyl. In certain embodiments, R.sub.3 is H, NH.sub.2, or N═CR.sub.4R.sub.6.
(41) In certain embodiments, each R.sub.4 is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl; each of which is optionally substituted.
(42) In certain embodiments, R.sub.6 is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted.
(43) In another aspect, the compound is a compound of formula IV:
(44) ##STR00004##
(45) wherein X is N or CR.sub.5; R.sub.5 is H, allyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; R.sub.B is H, alkyl, hydroxylalkyl, aminoalkyl, alkoxyalkyl, haloalkyl, hydroxy, alkoxy, or —COO—R.sub.3, each of which is optionally substituted; ring A is aryl or heteroaryl; each R.sub.A is independently alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or any two R.sub.A together with the atoms to which each is attached, can form a fused aryl or heteroaryl group; R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is H, —COO—R.sub.3, —CO—R.sub.3, —CO—N(R.sub.3R.sub.4), —S(O).sub.2—R.sub.3, —S(O).sub.2—N(R.sub.3R.sub.4), N(R.sub.3R.sub.4), N(R.sub.4)C(O)R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl; R.sub.2 is H, D, halogen, or optionally substituted alkyl; each R.sub.3 is independently selected from the group consisting of: (i) H, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; (ii) heterocycloalkyl or substituted heterocycloalkyl; (iii) —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl or —C.sub.2-C.sub.8 alkynyl, each containing 0, 1, 2, or 3 heteroatoms selected from O, S, or N; —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted; and (iv) NH.sub.2, N═CR.sub.4R.sub.6; each R.sub.4 is independently H, alkyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.3 and R.sub.4 are taken together with the nitrogen atom to which they are attached to form a 4-10-membered ring; R.sub.6 is alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, or heteroaryl, each of which is optionally substituted; or R.sub.4 and R.sub.6 are taken together with the carbon atom to which they are attached to form a 4-10-membered ring; m is 0, 1, 2, or 3; provided that (a) if ring A is thienyl, X is N, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0 and L is —CO—N(R.sub.3R.sub.4), then R.sub.3 and R.sub.4 are not taken together with the nitrogen atom to which they are attached to form a morpholino ring; (b) if ring A is thienyl, X is N, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0 and L is —CO—N(R.sub.2R.sub.4), and one of R.sub.3 and R.sub.4 is H, then the other of R.sub.3 and R.sub.4 is not methyl, hydroxyethyl, alkoxy, phenyl, substituted phenyl, pyridyl or substituted pyridyl; and (c) if ring A is thienyl, X is N, R.sub.2 is H, R.sub.B is methyl, and R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0 and L is —COO—R.sub.3, then R.sub.3 is not methyl or ethyl; or
a salt, solvate or hydrate thereof.
(46) In certain embodiments, R.sub.1 is —(CH.sub.2).sub.n-L, in which n is 0-3 and L is —COO—R.sub.3, optionally substituted aryl, or optionally substituted heteroaryl; and R.sub.3 is —C.sub.1-C.sub.8 alkyl, which contains 0, 1, 2, or 3 heteroatoms selected from O, S, or N, and which may be optionally substituted. In certain embodiments, n is 1 or 2 and L is alkyl or —COO—R.sub.3, and R.sub.3 is methyl, ethyl, propyl, i-propyl, butyl, sec-butyl, or t-butyl; or n is 1 or 2 and L is H or optionally substituted phenyl.
(47) In certain embodiments, R.sub.2 is H or methyl.
(48) In certain embodiments, R.sub.B is methyl, ethyl, hydroxy methyl, methoxymethyl, trifluoromethyl, COOH, COOMe, COOEt, COOCH.sub.2OC(O)CH.sub.3.
(49) In certain embodiments, ring A is phenyl, naphthyl, biphenyl, tetrahydronaphthyl, indanyl, pyridyl, furanyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl, imidazolyl, oxazolyl, thienyl, thiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, or 5,6,7,8-tetrahydroisoquinolinyl.
(50) In certain embodiments, each R.sub.A is independently an optionally substituted alkyl, or any two R.sub.A together with the atoms to which each is attached, can form an aryl.
(51) The methods of the invention also relate to compounds of Formulae V-XXII, and to any compound described herein.
(52) In another aspect, the compound is a compound represented by the formula:
(53) ##STR00005##
(54) or a salt, solvate or hydrate thereof.
(55) In certain embodiments, the compound is (+)-JQ1:
(56) ##STR00006##
(57) or a salt, solvate or hydrate thereof.
(58) In another aspect, the compound is a compound represented by the formula:
(59) ##STR00007##
(60) or a salt, solvate or hydrate thereof.
(61) In another aspect, the compound is a compound represented by the formula:
(62) ##STR00008##
or a salt, solvate or hydrate thereof.
(63) In another aspect, the compound is a compound represented by any one of the following formulae:
(64) ##STR00009## ##STR00010## ##STR00011##
or a salt, solvate or hydrate thereof.
(65) In another aspect, the compound is a compound represented by any one of the following formulae:
(66) ##STR00012##
(67) or a salt, solvate or hydrate thereof.
(68) In another aspect, the compound is a compound represented by any one of the following structures:
(69) ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019##
(70) or a salt, solvate or hydrate thereof.
(71) In certain embodiments, a compound of the invention can be represented by one of the following structures:
(72) ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024##
(73) or a salt, solvate or hydrate thereof.
(74) In one embodiment, the compound is represented by the structure:
(75) ##STR00025##
(76) or a salt, solvate or hydrate thereof.
(77) In another embodiment, the compound is represented by the structure:
(78) ##STR00026##
(79) or a salt, solvate or hydrate thereof.
(80) In another embodiment, the compound is represented by the structure:
(81) ##STR00027##
(82) or a salt, solvate or hydrate thereof.
(83) In certain embodiments, a compound of the invention can have the opposite chirality of any compound shown herein.
(84) In certain embodiments, the compound is a compound represented by Formula (V), (VI), or (VII):
(85) ##STR00028##
in which R, R.sub.1, and R.sub.2 and R.sub.B have the same meaning as in Formula (I); Y is O, N, S, or CR.sub.5, in which R.sub.5 has the same meaning as in Formula (I); n is 0 or 1; and the dashed circle in Formula (VII) indicates an aromatic or non-aromatic ring; or a salt, solvate, or hydrate thereof.
(86) In certain embodiments of any of the Formulae I-IV and VI (or any formula herein), R.sub.6 represents the non-carbonyl portion of an aldehyde shown in Table A, below (i.e., for an aldehyde of formula R.sub.6CHO, R.sub.6 is the non-carbonyl portion of the aldehyde). In certain embodiments, R.sub.4 and R.sub.6 together represent the non-carbonyl portion of a ketone shown in Table A (i.e., for a ketone of formula R.sub.6C(O)R.sub.4, R.sub.4 and R.sub.6 are the non-carbonyl portion of the ketone).
(87) TABLE-US-00005 TABLE A Plate 1 01 02 03 A
(88) In one embodiment, the compound is a compound is represented by the formula:
(89) ##STR00377##
or a salt, solvate, or hydrate thereof.
(90) In certain embodiments, the compound is (racemic) JQ1; in certain embodiments, the compound is (+)-JQ1. In certain embodiments, the compound is a compound selected from the group consisting of:
(91) ##STR00378##
or a salt, solvate, or hydrate thereof.
(92) Additional examples of compounds include compounds according to any of the follow formulae:
(93) ##STR00379## ##STR00380## ##STR00381##
(94) In Formulae IX-XXII, R and R′ can be, e.g., H, aryl, substituted aryl, heteroaryl, heteroaryl, heterocycloalkyl, —C.sub.1-C.sub.8 alkyl, —C.sub.2-C.sub.8 alkenyl, —C.sub.2-C.sub.8 alkynyl, —C.sub.3-C.sub.12 cycloalkyl, substituted —C.sub.3-C.sub.12 cycloalkyl, —C.sub.3-C.sub.12 cycloalkenyl, or substituted —C.sub.3-C.sub.12 cycloalkenyl, each of which may be optionally substituted. In Formulae XIV, X can be any substituent for an aryl group as described herein.
(95) Compounds of the invention can be prepared by a variety of methods, some of which are known in the art. For instance, the chemical Examples provided hereinbelow provide synthetic schemes for the preparation of the compound JQ1 (as the racemate) and the enantiomers (+)-JQ1 and (−)-JQ1 (see Schemes S1 and S2). A variety of compounds of Formulae (I)-(VIII) can be prepared by analogous methods with substitution of appropriate starting materials.
(96) For example, starting from JQ1, the analogous amine can be prepared as shown in Scheme 1, below.
(97) ##STR00382##
(98) As shown in Scheme 1, hydrolysis of the t-butyl ester of JQ1 affords the carboxylic acid, which is treated with diphenylphosphoryl azide (DPPA) and subjected to Curtius rearrangement conditions to provide the Cbz-protected amine, which is then deprotected to yield the amine. Subsequent elaboration of the amine group, e.g., by reductive amination yields secondary amines, which can be further alkylated to provide tertiary amines.
(99) ##STR00383##
(100) Scheme 2 shows the synthesis of further examples of the compounds of the invention, e.g., of Formula I, in which the fused ring core is modified (e.g., by substitution of a different aromatic ring as Ring A in Formula I). Use of aminodiarylketones having appropriate functionality (e.g., in place of the aminodiarylketone S2 in Scheme S1, infra) provides new compounds having a variety of fused ring cores and/or aryl group appendages (corresponding to group R in Formula I). Such aminodiarylketones are commercially available or can be prepared by a variety of methods, some of which are known in the art.
(101) Scheme 3 provides additional exemplary synthetic schemes for preparing further compounds of the invention.
(102) ##STR00384##
(103) As shown in Scheme 3, a fused bicyclic precursor (see Scheme S1, infra, for synthesis of this compound) is functionalized with a moiety R (DAM=dimethylaminomethylene protecting group) and then elaborated by reaction with a hydrazide to form the tricyclic fused core. Substituent R.sub.x can be varied by selection of a suitable hydrazide.
(104) Additional examples of compounds of the invention (which can be prepared by the methods described herein) include:
(105) Amides:
(106) Amides can be prepared, e.g., by preparation of a corresponding carboxylic acid or ester, followed by amidation with an appropriate amine using standard conditions. In certain embodiments, an amide provides a two-carbon “linker” with a terminal nitrogen-containing ring (e.g., pyridyl, piperidyl, piperazinyl, imidazolyl (including N-methyl-imidazolyl), morpholinyl, and the like. Exemplary amide structures include:
(107) ##STR00385## ##STR00386##
(108) The use of a two-carbon linker between the amide moiety and the terminal nitrogen-containing ring is preferred.
(109) “Reverse Amides”:
(110) ##STR00387## ##STR00388##
(111) Secondary Amines:
(112) ##STR00389## ##STR00390##
(113) Boronic Acids:
(114) ##STR00391##
(115) In certain embodiments, a compound having at least one chiral center is present in racemic form. In certain embodiments, a compound having at least one chiral center is enantiomerically enriched, i.e., has an enantiomeric excess (e.e.) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 90%, 95%, 99%, 99% or 100%. In certain embodiments, a compound has the same absolute configuration as the compound (+)-JQ1 ((S)-tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate) described herein.
(116) In certain embodiments of any of the Formulae disclosed herein, the compound is not represented by the following structure:
(117) ##STR00392##
(118) in which:
(119) R′.sub.1 is C.sub.1-C.sub.4 alkyl;
(120) R′.sub.2 is hydrogen, halogen, or C.sub.1-C.sub.4 alkyl optionally substituted with a halogen atom or a hydroxyl group;
(121) R′.sub.3 is a halogen atom, phenyl optionally substituted by a halogen atom, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, or cyano; —NR.sub.5—(CH.sub.2).sub.m—R.sub.6 wherein R.sub.5 is a hydrogen atom or C.sub.1-C.sub.4 alkyl, m is an integer of 0-4, and R.sub.6 is phenyl or pyridyl optionally substituted by a halogen atom; or —NR.sub.7—CO—(CH.sub.2).sub.n—R.sub.8 wherein R.sub.7 is a hydrogen atom or C.sub.1-C.sub.4 alkyl, n is an integer of 0-2, and R.sub.8 is phenyl or pyridyl optionally substituted by a halogen atom; and
(122) R′.sub.4 is —(CH.sub.2).sub.a—CO—NH—R.sub.9 wherein a is an integer of 1-4, and R.sub.9 is C.sub.1-C.sub.4 alkyl; C.sub.1-C.sub.4 hydroxyalkyl; C.sub.1-C.sub.4 alkoxy; or phenyl or pyridyl optionally substituted by C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, amino or a hydroxyl group or —(CH.sub.2).sub.b—COOR.sub.10 wherein b is an integer of 1-4, and R.sub.10 is C.sub.1-C.sub.4 alkyl.
(123) The term “pharmaceutically acceptable salt” also refers to a salt prepared from a compound disclosed herein (e.g., JQ1, a compound of Formulas I-XXII) or any other compound delineated herein, having an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic, or organic base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)-amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” also refers to a salt prepared from a compound disclosed herein, or any other compound delineated herein, having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include, but are not limited to, hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, glucosic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
(124) Methods of the Invention
(125) The present invention also relates to using the novel compounds described herein, as well as other inhibitors of BRDT as male contraceptives. Such compounds are known in the art and described, for example, in WO2009084693 or corresponding US2010286127.
(126) Thus, in one aspect, the invention provides methods for reducing or inhibiting spermatozoa emission involving administering an effective amount of a BRDT inhibitor to a male subject. In embodiments, the inhibitor is a compound having a formula delineated herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof.
(127) In embodiments, the methods involve administering the inhibitor in an amount sufficient to suppress spermatogenesis.
(128) In embodiments, the methods involve administering the inhibitor in an amount sufficient to induce azoospermia or oligozoospermia.
(129) In embodiments, the methods involve administering the inhibitor in an amount sufficient to lower the spermatozoa concentration to not more than 3 million/mL, 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, the methods involve administering the inhibitor in an amount sufficient to lower the spermatozoa concentration to not more than 0.1 million/mL.
(130) In embodiments, the inhibitor is administered in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
(131) In embodiments, the inhibitor is administered to the subject orally, transdermally, or by injection. In related embodiments, the inhibitor is administered in the form of a tablet or capsule. In related embodiments, the inhibitor is administered by parenteral injection, intramuscular injection, intravenous injection, subcutaneous implantation, subcutaneous injection, or transdermal preparation.
(132) In embodiments, the inhibitor is used in combination with at least one additional male contraceptive agent or device. In related embodiments, the additional male contraceptive is a condom. In other related embodiments, the additional male contraceptive is a modulator of testosterone production, androgen receptor function or stability.
(133) Pharmaceutical Compositions
(134) The invention features pharmaceutical compositions that contain one or more of the compounds described herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof as the active ingredient(s). The pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. These compositions can be useful as a male contraceptive.
(135) A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of the pharmaceutical composition should suit the mode of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans, and can be sterile, non-particulate and/or non-pyrogenic.
(136) Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited, to saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.
(137) Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the compositions.
(138) Examples of pharmaceutically-acceptable antioxidants include, but are not limited to: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
(139) In embodiments, the pharmaceutical composition is provided in a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
(140) In embodiments, the pharmaceutical composition is supplied in liquid form, for example, in a sealed container indicating the quantity and concentration of the active ingredient in the pharmaceutical composition. In related embodiments, the liquid form of the pharmaceutical composition is supplied in a hermetically sealed container.
(141) Methods for formulating the pharmaceutical compositions of the present invention are conventional and well-known in the art (see Remington and Remington's). One of skill in the art can readily formulate a pharmaceutical composition having the desired characteristics (e.g., route of administration, biosafety, and release profile).
(142) Methods for preparing the pharmaceutical compositions include the step of bringing into association the active ingredient with a pharmaceutically acceptable carrier and, optionally, one or more accessory ingredients. The pharmaceutical compositions can be prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Additional methodology for preparing the pharmaceutical compositions, including the preparation of multilayer dosage forms, are described in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th ed., Lippincott Williams & Wilkins), which is hereby incorporated by reference.
(143) Methods of Delivery
(144) The pharmaceutical compositions of the present invention can be administered to a subject by oral and non-oral means (e.g., topically, transdermally, or by injection). Such modes of administration and the methods for preparing an appropriate pharmaceutical composition for use therein are described in Gibaldi's Drug Delivery Systems in Pharmaceutical Care (1st ed., American Society of Health-System Pharmacists), which is hereby incorporated by reference.
(145) In embodiments, the pharmaceutical compositions are administered orally in a solid form.
(146) Pharmaceutical compositions suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) described herein, a derivative thereof, or a pharmaceutically acceptable salt or prodrug thereof as the active ingredient(s). The active ingredient can also be administered as a bolus, electuary, or paste.
(147) In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard-filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
(148) A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-actives, and/or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
(149) The tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the art.
(150) The pharmaceutical compositions can also be formulated so as to provide slow, extended, or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. The pharmaceutical compositions can also optionally contain opacifying agents and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more pharmaceutically acceptable carriers, excipients, or diluents well-known in the art (see, e.g., Remington and Remington's).
(151) The pharmaceutical compositions can be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
(152) In embodiments, the pharmaceutical compositions are administered orally in a liquid form.
(153) Liquid dosage forms for oral administration of an active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the liquid pharmaceutical compositions can include adjuvants such as wetting agents; emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents, and the like.
(154) Suspensions, in addition to the active ingredient(s) can contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
(155) In embodiments, the pharmaceutical compositions are administered by non-oral means such as by topical application, transdermal application, injection, and the like. In related embodiments, the pharmaceutical compositions are administered parenterally by injection, infusion, or implantation (e.g., intravenous, intramuscular, intraarticular, subcutaneous, and the like).
(156) Compositions for parenteral use can be presented in unit dosage forms, e.g. in ampoules or in vials containing several doses, and in which a suitable preservative can be added. Such compositions can be in form of a solution, a suspension, an emulsion, an infusion device, a delivery device for implantation, or it can be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. One or more co-vehicles, such as ethanol, can also be employed. Apart from the active ingredient(s), the compositions can contain suitable parenterally acceptable carriers and/or excipients or the active ingredient(s) can be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the compositions can also contain suspending, solubilising stabilising, pH-adjusting agents, and/or dispersing agents.
(157) The pharmaceutical compositions can be in the form of sterile injections. To prepare such a composition, the active ingredient is dissolved or suspended in a parenterally acceptable liquid vehicle. Exemplary vehicles and solvents include, but are not limited to, water, water adjusted to a suitable by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The pharmaceutical composition can also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate. To improve solubility, a dissolution enhancing or solubilising agent can be added or the solvent can contain 10-60% w/w of propylene glycol or the like.
(158) The pharmaceutical compositions can contain one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted into sterile injectable solutions or dispersions just prior to use. Such pharmaceutical compositions can contain antioxidants; buffers; bacteriostats; solutes, which render the formulation isotonic with the blood of the intended recipient; suspending agents; thickening agents; preservatives; and the like.
(159) Examples of suitable aqueous and nonaqueous carriers, which can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
(160) In some embodiments, in order to prolong the effect of an active ingredient, it is desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the active ingredient then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered active ingredient is accomplished by dissolving or suspending the compound in an oil vehicle. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
(161) Controlled release parenteral compositions can be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants or infusion devices.
(162) Materials for use in the preparation of microspheres and/or microcapsules include biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).
(163) Biocompatible carriers which can be used when formulating a controlled release parenteral formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies.
(164) Materials for use in implants can be non-biodegradable, e.g., polydimethylsiloxane, or biodegradable such as, e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters).
(165) In embodiments, the active ingredient(s) are administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension can be used. The pharmaceutical composition can also be administered using a sonic nebulizer, which would minimize exposing the agent to shear, which can result in degradation of the compound.
(166) Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the active ingredient(s) together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
(167) Dosage forms for topical or transdermal administration of an active ingredient(s) includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient(s) can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants as appropriate.
(168) Transdermal patches suitable for use in the present invention are disclosed in Transdermal Drug Delivery: Developmental Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S. Pat. Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, which are hereby incorporated by reference. The transdermal patch can also be any transdermal patch well-known in the art, including transscrotal patches. Pharmaceutical compositions in such transdermal patches can contain one or more absorption enhancers or skin permeation enhancers well-known in the art (see, e.g., U.S. Pat. Nos. 4,379,454 and 4,973,468, which are hereby incorporated by reference). Transdermal therapeutic systems for use in the present invention can be based on iontophoresis, diffusion, or a combination of these two effects.
(169) Transdermal patches have the added advantage of providing controlled delivery of active ingredient(s) to the body. Such dosage forms can be made by dissolving or dispersing the active ingredient(s) in a proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient(s) in a polymer matrix or gel.
(170) Such pharmaceutical compositions can be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other kinds of transdermal drug delivery systems. The compositions can also include pharmaceutically acceptable carriers or excipients such as emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents, ointment bases, perfumes, and skin protective agents.
(171) Examples of emulsifying agents include, but are not limited to, naturally occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and sorbitan monooleate derivatives.
(172) Examples of antioxidants include, but are not limited to, butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, and cysteine.
(173) Examples of preservatives include, but are not limited to, parabens, such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.
(174) Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol and urea.
(175) Examples of penetration enhancers include, but are not limited to, propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycol monoethyl or monomethyl ether with propylene glycol monolaurate or methyl laurate, eucalyptol, lecithin, Transcutol®, and Azone®.
(176) Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid and phosphoric acid.
(177) Examples of gel forming agents include, but are not limited to, Carbopol, cellulose derivatives, bentonite, alginates, gelatin and polyvinylpyrrolidone.
(178) In addition to the active ingredient(s), the ointments, pastes, creams, and gels of the present invention can contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
(179) Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.
(180) Injectable depot forms are made by forming microencapsule matrices of compound(s) of the invention in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer, and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
(181) Subcutaneous implants are well-known in the art and are suitable for use in the present invention. Subcutaneous implantation methods are preferably non-irritating and mechanically resilient. The implants can be of matrix type, of reservoir type, or hybrids thereof. In matrix type devices, the carrier material can be porous or non-porous, solid or semi-solid, and permeable or impermeable to the active compound or compounds. The carrier material can be biodegradable or may slowly erode after administration. In some instances, the matrix is non-degradable but instead relies on the diffusion of the active compound through the matrix for the carrier material to degrade. Alternative subcutaneous implant methods utilize reservoir devices where the active compound or compounds are surrounded by a rate controlling membrane, e.g., a membrane independent of component concentration (possessing zero-order kinetics). Devices consisting of a matrix surrounded by a rate controlling membrane also suitable for use.
(182) Both reservoir and matrix type devices can contain materials such as polydimethylsiloxane, such as Silastic™, or other silicone rubbers. Matrix materials can be insoluble polypropylene, polyethylene, polyvinyl chloride, ethylvinyl acetate, polystyrene and polymethacrylate, well as glycerol esters of the glycerol palmitostearate, glycerol stearate, and glycerol behenate type. Materials can be hydrophobic or hydrophilic polymers and optionally contain solubilising agents.
(183) Subcutaneous implant devices can be slow-release capsules made with any suitable polymer, e.g., as described in U.S. Pat. Nos. 5,035,891 and 4,210,644, which are hereby incorporated by reference.
(184) In general, at least four different approaches are applicable in order to provide rate control over the release and transdermal permeation of a drug compound. These approaches are: membrane-moderated systems, adhesive diffusion-controlled systems, matrix dispersion-type systems and microreservoir systems. It is appreciated that a controlled release percutaneous and/or topical composition can be obtained by using a suitable mixture of these approaches.
(185) In a membrane-moderated system, the active ingredient is present in a reservoir which is totally encapsulated in a shallow compartment molded from a drug-impermeable laminate, such as a metallic plastic laminate, and a rate-controlling polymeric membrane such as a microporous or a nonporous polymeric membrane, e.g., ethylene-vinyl acetate copolymer. The active ingredient is released through the ratecontrolling polymeric membrane. In the drug reservoir, the active ingredient can either be dispersed in a solid polymer matrix or suspended in an unleachable, viscous liquid medium such as silicone fluid. On the external surface of the polymeric membrane, a thin layer of an adhesive polymer is applied to achieve an intimate contact of the transdermal system with the skin surface. The adhesive polymer is preferably a polymer which is hypoallergenic and compatible with the active drug substance.
(186) In an adhesive diffusion-controlled system, a reservoir of the active ingredient is formed by directly dispersing the active ingredient in an adhesive polymer and then by, e.g., solvent casting, spreading the adhesive containing the active ingredient ance onto a flat sheet of substantially drug-impermeable metallic plastic backing to form a thin drug reservoir layer.
(187) A matrix dispersion-type system is characterized in that a reservoir of the active ingredient is formed by substantially homogeneously dispersing the active ingredient in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into disc with a substantially well-defined surface area and controlled thickness. The adhesive polymer is spread along the circumference to form a strip of adhesive around the disc.
(188) A microreservoir system can be considered as a combination of the reservoir and matrix dispersion type systems. In this case, the reservoir of the active substance is formed by first suspending the drug solids in an aqueous solution of water-soluble polymer and then dispersing the drug suspension in a lipophilic polymer to form a multiplicity of unleachable, microscopic spheres of drug reservoirs.
(189) Any of the above-described controlled release, extended release, and sustained release compositions can be formulated to release the active ingredient in about 30 minutes to about 1 week, in about 30 minutes to about 72 hours, in about 30 minutes to 24 hours, in about 30 minutes to 12 hours, in about 30 minutes to 6 hours, in about 30 minutes to 4 hours, and in about 3 hours to 10 hours. In embodiments, an effective concentration of the active ingredient(s) is sustained in a subject for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, or more after administration of the pharmaceutical compositions to the subject.
(190) Methods of Delivery
(191) When the compound(s) of the invention are administered as pharmaceuticals to humans and animals, they can be given per se or as a pharmaceutical composition containing active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.
(192) Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. Generally, compounds or pharmaceutical compositions of the invention are administered in an effective amount or quantity sufficient to reduce or inhibit spermatozoa emission in a male subject. In embodiments, administration of the compound or pharmaceutical composition suppresses spermatogenesis, induces azoospermia, or induces oligozoospermia.
(193) Exemplary dose ranges include 0.01 mg to 250 mg per day, 0.01 mg to 100 mg per day, 1 mg to 100 mg per day, 10 mg to 100 mg per day, 1 mg to 10 mg per day, and 0.01 mg to 10 mg per day. A preferred dose of the compound of the invention is the maximum that a patient can tolerate and not develop serious or unacceptable side effects. In embodiments, the compound(s) of the present invention is administered at a concentration of about 10 micrograms to about 100 mg per kilogram of body weight per day, about 0.1 to about 10 mg/kg per day, or about 1.0 mg to about 10 mg/kg of body weight per day.
(194) In embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount ranging between 1 and 10 mg, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg.
(195) In embodiments, the therapeutically effective dosage produces a serum concentration of compound of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. For example, dosages for systemic administration to a human patient can range from 1-10 μg/kg, 20-80 μg/kg, 5-50 μg/kg, 75-150 μg/kg, 100-500 μg/kg, 250-750 μg/kg, 500-1000 μg/kg, 1-10 mg/kg, 5-50 mg/kg, 25-75 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 50-100 mg/kg, 250-500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000 mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg, 1000 mg/kg, 1500 mg/kg, or 2000 mg/kg. Pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 5000 mg, for example from about 100 to about 2500 mg of the compound or a combination of essential ingredients per dosage unit form.
(196) In embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount sufficient to lower spermatozoa concentration to not more than 3 million/mL of semen, such as not more than 2 million/mL, 1 million/mL, 0.5 million/mL, 0.25 million/mL, or 0.1 million/mL. In related embodiments, the pharmaceutical composition comprises a compound(s) of the invention in an amount sufficient to lower spermatozoa concentration to not more than 0.1 million/mL.
(197) Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an efficacious or effective amount of a compound(s) of the invention is determined by first administering a low dose of the compound(s) and then incrementally increasing the administered dose or dosages until a desired effect (e.g., decreased spermatozoa levels in seminal fluid) is observed in the treated subject, with minimal or acceptable toxic side effects. Applicable methods for determining an appropriate dose and dosing schedule for administration of a pharmaceutical composition of the present invention are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott & Wilkins (2003 and 2005), which are hereby incorporated by reference.
(198) Kits
(199) The invention provides for a kit for effecting male contraception. In embodiments, the kit contains one or more of the compounds or pharmaceutical compositions described herein. In embodiments, the kit provides instructions for use. The instructions for use can pertain to any of the methods described herein. In related embodiments, the instructions pertain to using the compound(s) or pharmaceutical composition(s) for reducing or inhibiting spermatozoa emission. In embodiments, the kit provides a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale of the kit and the components therein for human administration.
(200) The invention also provides for compound(s) or pharmaceutical composition(s) packaged in a hermetically sealed container (e.g., ampoule or sachette) indicating the quantity of compound. In embodiments, a compound or pharmaceutical composition is supplied as a liquid. In other embodiments, a compound or pharmaceutical composition is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline, to the appropriate concentration for administration to a subject.
(201) The invention also provides for transdermal patches containing the compound(s) or pharmaceutical composition(s).
(202) In embodiments, the kit provides compound(s) or pharmaceutical composition(s) in more than one dosage unit. The kit can contain from 1 to about 120 or more, from 1 to about 60, from 1 to about 30, from 1 to about 10, or from 1 to about 7 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to release a therapeutically effective amount of the active ingredient over a 24 hour period, the kit conveniently comprises 1, about 5, about 7, about 10, about 14, or about 30 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to provide a therapeutically effective amount of the active ingredient over a 12 hour period, the kit conveniently comprises 1, 2, about 10, about 14, about 30 or about 60 dosage units. In cases where the compound(s) or pharmaceutical composition(s) is adapted to provide a therapeutically effective amount of the active ingredient over an about 3 to about 10 hour (e.g., about a 6 or 8 hour) period, the kit comprises about 1, about 4, about 40, about 60 or about 120 dosage units. One skilled in the art will recognize that other numbers of dosage units can be included in the kit without departing materially from the present invention.
(203) Screening Methods
(204) As described herein, the invention provides specific examples of chemical compounds, including JQ1, as well as other substituted compounds that bind a bromodomain binding pocket and are useful as a male contraceptive. However, the invention is not so limited. The invention further provides a simple means for identifying agents (including nucleic acids, peptides, small molecule inhibitors, and mimetics) that are capable of inhibiting spermatogenesis. Such compounds are also expected to be useful as male contraceptives.
(205) In particular embodiments, the effect of a compound or other agent of the invention is analyzed by assaying spermatogenesis. Agents and compounds of the invention that reduce spermatogenesis are identified as useful as male contraceptives.
(206) Virtually any agent that specifically binds to a BET family member or that reduces the biological activity of a BET family member may be employed in the methods of the invention. Methods of the invention are useful for the high-throughput low-cost screening of candidate agents that reduce or otherwise inhibit spermatogenesis. A candidate agent that specifically binds to a bromodomain of a BET family member is then isolated and tested for activity in an in vitro assay or in vivo assay for its ability to inhibit spermatogenesis. One skilled in the art appreciates that the effects of a candidate agent on a cell is typically compared to a corresponding control cell not contacted with the candidate agent. Thus, the screening methods include comparing spermatogenesis in a testes contacted by a candidate agent to the spermatogenesis present in an untreated control testes.
(207) Once identified, agents of the invention (e.g., agents that specifically bind to and/or antagonize a bromodomain) may be used as male contraceptives. Potential bromodomain antagonists include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid ligands, aptamers, and antibodies that bind to a BET family member bromodomain and reduce its activity. Candidate agents may be tested for their ability to reduce spermatogenesis.
(208) Test Compounds and Extracts
(209) In certain embodiments, BET family member antagonists (e.g., agents that specifically bind and reduce the activity of a bromodomain) are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts or chemical libraries or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Virtually any number of unknown chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as the modification of existing polypeptides.
(210) Libraries of natural polypeptides in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Such polypeptides can be modified to include a protein transduction domain using methods known in the art and described herein. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; Erb et al., Proc. Natl. Acad. Sci. USA 91:11422, 1994; Zuckermann et al., J. Med. Chem. 37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. 37:1233, 1994. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
(211) Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of polypeptides, chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, chemical compounds to be used as candidate compounds can be synthesized from readily available starting materials using standard synthetic techniques and methodologies known to those of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds identified by the methods described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
(212) Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990; Cwirla et al. Proc. Natl. Acad. Sci. 87:6378-6382, 1990; Felici, J. Mol. Biol. 222:301-310, 1991; Ladner supra).
(213) In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their activity should be employed whenever possible.
(214) When a crude extract is found to have BET family member bromodomain binding activity further fractionation of the positive lead extract is necessary to isolate molecular constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract that reduces spermatogenesis. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful as therapeutics are chemically modified according to methods known in the art.
EXAMPLES
(215) It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
I. Chemical Examples
Synthesis and Methods of Preparation
(216) Compounds of the invention can be synthesized by methods described herein, and/or according to methods known to one of ordinary skill in the art in view of the description herein.
(217) ##STR00393## ##STR00394##
(2-amino-4,5-dimethylthiophen-3-yl)(4-chlorophenyl)methanone (S2)
(218) The compound JQ1 was prepared according to the scheme shown above.
(219) Sulfur (220 mg, 6.9 mmol, 1.00 equiv) was added as a solid to a solution of 4-chlorobenzoyl acetonitrile S1 (1.24 g, 6.9 mmol, 1 equiv), 2-butanone (0.62 ml, 6.9 mmol, 1.00 equiv), and morpholine (0.60 ml, 6.9 mmol, 1.00 equiv) in ethanol (20 ml, 0.35 M) at 23° C..sup.21. The mixture was then heated to 70° C. After 12 hours, the reaction mixture was cooled to 23° C. and poured into brine (100 ml). The aqueous layer was extracted with ethyl acetate (3×50 ml). The combined organic layers were washed with brine (50 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S2 (1.28 g, 70%) as a yellow solid.
(S)-tert-Butyl-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-{[3-(4-chorobenzoyl)-4,5-dimethylthiophen-2-yl]amino}-4-oxobutanoate (S3)
(220) (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) (827 mg, 2.0 mmol, 2.00 equiv), and N,N-diisopropylethylamine (0.72 ml, 4.0 mmol, 4.00 equiv) were added sequentially to a solution of 9-fluorenylmethoxycarbonyl-aspartic acid β-tert-butyl ester [Fmoc-Asp(Ot-Bu)-OH] (864 mg, 2.1 mmol, 2.10 equiv) in N,N-dimethylformamide (1.5 ml, 1.0 M). The mixture was then stirred at 23° C. for 5 min. S2 (266 mg, 1.0 mmol, 1 equiv) was then added as a solid. The reaction mixture was stirred at 23° C. After 16 hours, ethyl acetate (20 ml) and brine (20 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (30 ml), were dried over with anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S3 (625 mg, 90%) as brown oil.
(S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-oxobutanoate (S4)
(221) Compound S3 (560 mg, 0.85 mmol, 1 equiv) was dissolved into 20% piperidine in DMF solution (4.0 ml, 0.22 M) at 23° C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (3×25 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford free amine S4 (370 mg, 90%) as yellow solid. The enantiomeric purity dropped to 75% (determined with Berger Supercritical Fluid Chromatography (SFC) using AS-H column).
(S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (S5)
(222) Amino ketone (S4) (280 mg, 0.63 mmol) was dissolved in 10% acetic acid ethanol solution (21 ml, 0.03 M). The reaction mixture was heated to 85° C. After 30 minutes, all solvents were removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 12 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford compound S5 (241 mg, 95%) as white solid. Enantiomeric purity of S5 was 67% (determined with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).
tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-thioxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (S6)
(223) Phosphorus pentasulfide (222 mg, 1.0 mmol, 2.00 equiv), sodium bicarbonate (168 mg, 2.0 mmol, 4.00 equiv) were added sequentially to a solution of S5 (210 mg, 0.5 mmol, 1 equiv) diglyme (1.25 ml, 0.4M). The reaction mixture was heated to 90° C. After 16 h, brine (20 ml) and ethyl acetate (35 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (3×30 ml). The combined organic layers were washed with brine (2×15 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S6 (141 mg, 65%) as brown solid with recovered S5 (73 mg, 34%).
tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate [(±)JQ1]
(224) Hydrazine (0.015 ml, 0.45 mmol, 1.25 equiv) was added to a solution of S6 (158 mg, 0.36 mmol, 1 equiv) in THF (2.6 ml, 0.14 M) at 0° C. The reaction mixture was warmed to 23° C., and stirred at 23° C. for 1 h. All solvents were removed under reduced pressure. The resulting hydrazine was used directly without purification. The hydrazine was then dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (6 ml, 0.06 M). The reaction mixture was heated to 120° C. After 2 h, all the solvents were removed under reduced pressure. The residue was purified by flash column chromatography (Combiflash system, 4 g silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford JQ1 (140 mg, 85% in 2 steps) as white solid. The reaction conditions further epimerized the stereogenic center, resulting in the racemate, JQ1 (determined with Berger Supercritical Fluid Chromatography (SFC) with an AS-H column).
(225) ##STR00395##
(S)-tert-Butyl-3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-4-{[3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl]amino}-4-oxobutanoate (S3)
(226) (Benzotriazol-1-yloxyl)tripyrrolidinophosphonium (PyBOP) (494 mg, 0.95 mmol, 0.95 equiv), N,N-diisopropylethylamine (0.50 ml, 2.8 mmol, 2.75 equiv) were added sequentially to a solution of 9-fluorenylmethoxycarbonyl-aspartic acid p-tert-butyl ester [Fmoc-Asp(Ot-Bu)-OH] (411 mg, 1.00 mmol, 1.0 equiv) in N,N-dimethylformamide (1.0 ml, 1.0 M). The mixture was then stirred at 23° C. for 5 min. S2 (266 mg, 1.0 mmol, 1 equiv) was then added as solid. The reaction mixture was stirred at 23° C. After 4 h, ethyl acetate (20 ml) and brine (20 ml) were added. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine, were dried over with anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 40 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford S3 (152 mg, 72%) as brown oil.
(S)-tert-butyl 3-amino-4-((3-(4-chlorobenzoyl)-4,5-dimethylthiophen-2-yl)amino)-4-oxobutanoate (S4)
(227) Compound S3 (310 mg, 0.47 mmol, 1 equiv) was dissolved into 20% piperidine in DMF solution (2.2 ml, 0.22 M) at 23° C. After 30 min, ethyl acetate (20 ml) and brine (20 ml) were added to the reaction mixture. The two layers were separated, and the aqueous layer was extracted with ethyl acetate (2×20 ml). The combined organic layers were washed with brine (3×25 ml), were dried over anhydrous sodium sulphate, were filtered, and were concentrated under reduced pressure. The residue was purified by flash column chromatography (Combiflash RF system, 24 gram silica gel, gradient 0 to 100% ethyl acetate-hexane) to afford free amine S4 (184 mg, 90%) as yellow solid. The enantiomeric purity was 91% (checked with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).
(S)-tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2-oxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (S5)
(228) Amino ketone (S4) (184 mg, 0.42 mmol) was dissolved in toluene (10 ml, 0.04 M). Silica gel (300 mg) was added, and the reaction mixture was heated to 90° C. After 3 h, the reaction mixture was cooled to 23° C. The silica gel was filtered, and washed with ethyl acetate. The combined filtrates were concentrated. The residue was purified by flash column chromatography (Combiflash RF system, 12 gram silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford compound S5 (168 mg, 95%) as white solid. Enantiomeric purity of S5 was 90% (determined with Berger Supercritical Fluid Chromatography (SFC) using an AS-H column).
(S)-tert-Butyl 2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate [(+)JQ1]
(229) Potassium tert-butoxide (1.0 M solution in THF, 0.3 ml, 0.30 mmol, 1.10 equiv) was added to a solution of S5 (114 mg, 0.27 mmol, 1 equiv) in THF (1.8 ml, 0.15 M) at −78° C. The reaction mixture was warmed to −10° C., and stirred at 23° C. for 30 min. The reaction mixture was cooled to −78° C. Diethyl chlorophosphate (0.047 ml, 0.32 mmol, 1.20 equiv) was added to reaction mixture.sup.22. The resulting mixture was warmed to −10° C. over 45 min. Acetic hydrazide (30 mg, 0.40 mmol, 1.50 equiv) was added to reaction mixture. The reaction mixture was stirred at 23° C. After 1 h, 1-butanol (2.25 ml) was added to reaction mixture, which was heated to 90° C. After 1 h, all solvents were removed under reduce pressure. The residue was purified with flash column chromatography (Combiflash system, 4 g silica gel, gradient 0 to 100% ethyl acetate-hexanes) to afford (+)-JQ1 (114 mg, 92%) as white solid with 90% enantiomeric purity (determined with Berger Supercritical Fluid Chromatography (SFC) using AS-H column, 85% hexanes-methanol, 210 nm, t.sub.R (R-enantiomer)=1.59 min, t.sub.R (S-enantiomer)=3.67 min). The product was further purified by chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to provide the S-enantiomer in greater than 99% ee.
(230) .sup.1H NMR (600 MHz, CDCl.sub.3, 25° C.) δ 7.39 (d, J=8.4 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 4.54 (t, J=6.6 MHz, 1H), 3.54-3.52 (m, 2H), 2.66 (s, 3H), 2.39 (s, 3H), 1.67 (s, 3H), 1.48 (s, 9H).
(231) .sup.13C NMR (150 MHz, CDCl.sub.3, 25° C.) δ 171.0, 163.8, 155.7, 150.0, 136.9, 131.1, 130.9, 130.6, 130.3, 128.9, 81.2, 54.1, 38.1, 28.4, 14.6, 13.5, 12.1.
(232) HRMS(ESI) calc'd for C.sub.21H.sub.24ClN.sub.2O.sub.3S [M+H].sup.+: 457.1460, found 457.1451 m/z.
(233) TLC (EtOAc), Rf: 0.32 (UV)
(234) [α].sup.22.sub.D=+75 (c 0.5, CHCl.sub.3)
(235) (−)-JQ1 was synthesized in a similar manner, employing Fmoc-D-Asp(Ot-Bu)-OH as a starting material, and was further purified by chiral preparative HPLC (Agilent High Pressure Liquid Chromatography using an OD-H column) to afford the R-enantiomer in greater than 99% ee. [α].sup.22.sub.D=−72 (c 0.5, CHCl.sub.3)
(236) Synthesis of Additional Compounds
(237) Additional compounds of the invention were prepared as illustrated in Scheme S3.
(238) ##STR00396##
(239) As shown in Scheme S3, the t-butyl ester of (+)-JQ1 (1) was cleaved to yield the free acid (2), which was coupled with hydrazine to yield the hydrazide (3). Reaction with 4-hydroxybenzaldehyde yielded the hydrazone (4).
(240) Both hydrazide (3) and hydrazone (4) showed activity in at least one biological assay.
(241) A library of compounds was prepared by reaction of the hydrazide (3) with a variety of carbonyl-containing compounds (see Table A, above).
(242) Additional compounds were prepared for use, e.g., as probes for assay development. An exemplary synthesis is shown in Scheme S4, below.
(243) ##STR00397## ##STR00398##
(244) Additional compounds were prepared as shown in the table below:
(245) TABLE-US-00006 Compound MS [M + H].sup.+ Name Structure m/z (Observed) (S)-JQ1
Spectral data for each compound were consistent with the assigned structure.
II. Biological Activity and Methods of Treatment
Example 1
JQ1 is an Inhibitor of BRDT
(246) The feasibility of targeting human bromodomains with acetyl-lysine competitive small molecules was recently established (Filippakopoulos et al., Nature 468:1067 (2010)). The index study identified a potent thienodiazepine inhibitor ((+)-JQ1;
(247) To assess competitive binding to BRDT(1), a homogeneous, luminescence proximity assay (alpha-screen), capable of quantifying binding of a synthetic, biotinylated tetra-acetylated histone 4 peptide (H4Kac4, residues 1-20) to recombinant epitope-tagged BRDT(1) was employed. Dose-ranging studies of (+)-JQ1 demonstrated potent inhibition of H4Kac4 binding, with a half-maximum inhibitory concentration (IC.sub.50) value of 11 nM (
Example 2
JQ1 Inhibits BRDT Activity During Spermatogenesis
(248) To determine the possible consequences of blocking BRDT function in vivo, the spermatogenic effects of JQ1 administered to male mice were evaluated. Murine BRDT(1) exhibits 90% amino acid sequence identity and 95% similarity to human BRDT(1), including all surface residues influencing molecular recognition (
(249) To further characterize these defects, spermatozoa number was determined after 3 weeks of treatment (3-6 weeks of age). It was found that epididymal sperm number were reduced to 27.8% of the control while after 6 weeks of treatment, the sperm in the cauda epididymis of the JQ1-treated mice were 10.9% of the control (
Example 3
JQ1 is a Reversible Inhibitor of BRDT Activity
(250) To further evaluate the consequences of JQ1 on male fertility and fertilization potential, control (n=2) and JQ1-treated (n=3) males treated for 3 weeks were housed with 2 females each and subjected to treatments for an additional 3 weeks. Whereas the control males impregnated all 4 females, JQ1 had a contraceptive effect on the males (one failed to impregnate the two females, whereas only 1 of 2 females in each of the other two cages became pregnant). When these same males were test bred to superovulated females (2 females per cage), after 5 weeks of treatment, all females demonstrated copulation plugs indicating that JQ1 did not alter mating behavior, consistent with normal testosterone-responsive tissues in these males. Oocytes from these females were collected from their oviductal ampulla and cultured for 2 days to determine their developmental potential post-mating (
Example 4
Molecular Analysis of JQ1 Mediated BRDT Inhibition
(251) To molecularly define the stages of spermatogenesis at which JQ1 functions, quantitative RT-PCR was performed on testes isolated from JQ1-treated mice and controls (
(252) A pharmacologic approach to male contraception remains a longstanding challenge in medicine. The results described herein provide pharmacologic validation of the amino-terminal bromodomain of BRDT as a target for male contraception, using a highly potent and selective chemical probe. JQ1 emerges as a lead compound for a new class of drugs that can cross the blood:testis boundary, inhibit bromodomain activity during spermatogenesis, impair sperm generation and motility, reduce the number of oocytes fertilized, and produce a reversible contraceptive effect in mammals. As human and mouse BRDT proteins are highly conserved and have nearly identical bromodomain pockets based on our structural predictions, these discoveries can be completely translated to men, and provide a novel and efficacious strategy for a male contraceptive.
(253) The results reported herein were obtained using the following methods and materials.
(254) (+)-JQ1
(255) The direct-acting, small-molecule bromodomain inhibitor was prepared as previously described (Filippakopoulos et al., Nature 468:1067 (2010)).
(256) Protein Cloning, Expression and Purification
(257) The N-terminal domain of human BRDT was cloned, expressed in E-Coli and purified as previously described (Filippakopoulos et al.).
(258) BRDT Proximity Assay
(259) Assays were performed with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5 and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions all subsequent steps were performed in low light conditions. A 2× solution of components with final concentrations of BRDT at 80 nM, Ni-coated Acceptor Bead at 25 μg/ml, and 80 nM biotinylated H4-tetra acetyl was added in 10 μL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Biotinylated peptide for BRDT was synthesized in-house on a CEM Liberty 9008005 microwave peptide synthesizer: H4-tetra acetyl, Biotin-PEG2-SGRGKacGGKacGLGKacGGAKacRHRK-COOH. Addition to wells was performed with either a multichannel pipet (for optimization experiments) or a Biotek EL406 liquid handler. After a 1 minute 1000 rpm spin-down, 100 nl of compounds from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 μg/ml final) were added as with previous solution in a 2×, 10 μl volume. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates were spun down again at 1000 rpm for 1 minute. Next, the plates were incubated in the room with the plate reader (for temperature equilibration) for 1.5 hour prior to reading the assay. AlphaScreen measurements were performed on an Envision 2104 (PerkinElmer, USA) utilizing the manufacturer's protocol.
(260) Sequence Alignment
(261) Amino acid sequences for full-length bromodomain-containing proteins were obtained from the US National Heart, Lung and Blood Institute (Human BRDT accession number Q58F21; Human BRD4 accession number O60885; Mouse BRDT accession number Q91Y44). Multiple sequence alignments of full-length BRDT and BRD4 were generated using MAFFT (v6.240) (Katoh et al., Nucleic Acids Res. 33:511 (2005); Katoh et al., Nucleic Acids Res. 30:3059 (2002); and Katoh and Toh, Brief Bioinform. 9:286 (2008)). The E-INS-i algorithm was selected as suitable for sequences containing potentially large unalignable regions, and the BLOSUM62 scoring matrix was used as suitable for highly evolutionarily conserved sequences. Gap opening penalty and offset value were set to default parameters.
(262) Mouse Studies
(263) (+)-JQ1 was dissolved in DMSO at 50 mg/ml and then diluted 1:10 in (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich, St. Louis, Mo.). The subsequent mixture was injected intraperitoneal into male mice at 1% of the body weight of the mouse (final amount is 50 mg/kg/day). The control was DMSO dissolved 1:10 in (2-Hydroxypropyl)-β-cyclodextrin and injected similarly. Juvenile or adult C57BL6/J/129S5 hybrid mice for these studies were weighed daily before injections and fed ad libitum. These studies were approved by the Administrative Committee on Laboratory Animal Care at Baylor College of Medicine, and all experiments were conducted in accordance with the NIH guide for the Care and Use of Laboratory Animals.
(264) Histological Analysis
(265) Histological analysis of Bouin's fixed testes and epididymides was performed as previously described (Kumar et al., Nature Genetics 15:201 (1997)) using Periodic acid-Schiff and hematoxylin. Rabbit anti-TNP2 (1:600) staining and hematoxylin counter-staining was performed as described (Zhao et al., Biol. Reprod. 71:1016 (2004)) using Bouin's fixed testes.
(266) Epididymal Sperm Counts
(267) Counts were performed on spermatozoa isolated from the entire epididymis or from the caudal epididymis of adult mice as described (Roy et al., Faseb J. 21:1013 (2007)). In brief, epididymides were dissected and placed in prewarmed M2 medium, minced, and incubated at 37° C. in a incubator prior to counting.
(268) Fertilization and Embryo Developmental Potential
(269) To evaluate the ability of spermatozoa of treated mice to mate with females and fertilize oocytes, 21-day-old C57BL6/J/129S5 hybrid females were injected with 5 IU of pregnant mare serum gonadotropin (PMSG; Calbiochem, EMD, Gibbstown, N.J.) followed by 5 IU of human chorionic gonadotropin (hCG; Calbiochem, EMD, Gibbstown, N.J.) 48 hours later and mated to treated males. Oocytes were isolated from ampullas of oviducts of females with copulation plugs, counted, and cultured in M16 medium (Sigma-Aldrich, St. Louis, Mo.) for 24 hours (for counting of 2 cell embryos) and 48 hours (for counting of 4 cell embryos) as described (Andreu-Vieyra et al., PLoS Biol. 8:e1000453 (2010); and Burns et al., Science 300:633 (2003)).
(270) Quantitative RT-PCR Analysis
(271) Total RNAs from mouse testes were isolated using TRizol reagent (Invitrogen, Carlsbad, Calif.). Total RNA was then reversely transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, Calif.). Quantitative PCR was performed using SYBR green master mix and customized primers (Table 1).
(272) TABLE-US-00007 TABLE 1 Primers for quantitative PCR Gene name Forward Reverse Plzf TGGAGAAGCATTTGGGTATCTACTC AAGACGGCATGCTCAACACA Stra8 GAGTGAGGCCCAGCATATGTC CCTCTGGATTTTCTGAGTTGCA Brdt GCTTTGGGACTCCACAACTACTATG GATTGTCCATTTTCCCCTTGATC Ccna1 TTTCCCCAATGCTGGTTGA AACCAAAATCCGTTGCTTCT His1hlt GCTGATTCCTGAGGCCCTTT CAGGGCAGCAAGGGACAT Papolb CGCCAACAGAGAAACAACATTTAG CCAACCAGGATTCGGATCTTT Klf17 CCTCCCGTTTGTTCTCAACTTG GGTGCATAGCCTGTTCCTTATTG Prm1 TGCACAGAATAGCAAGTCCATCA TGTGGCGAGATGCTCTTGAA
All quantitative PCR assays were conducted in duplicate for each sample. Gapdh was used as an internal control for the quantification.
Other Embodiments
(273) From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
(274) The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
(275) All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. The subject matter described herein may be related to subject matter of U.S. provisional applications 61/334,991, 61/370,745, and 61/375,663, each of which is incorporated herein by this reference.